Mouse Dominant Mutations

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Mouse Dominant Mutations
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Mouse recessive mutations
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Transgenic Mice
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Founders Germline Integration
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Complementation cloning
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Inducible expression of transgenes
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Mouse es cells
Once derived functional es cells can be
propagated stably
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Select
Check
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Positive (neo), Negative (TK) Selection
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Knock-in
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Mouse chimeras
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Genetic Mosaics
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Yeast Vectors
All have selectable markers (HIS5, TRP1, LEU2
etc.)
High copy, segregate
Plasmids based on 2 micron plasmid
Single copy, segregate
Centromere plasmids (small circular)
Single copy, segregate
YAC (long, linear)
Not replicated or segregated
Integrating Plasmids
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Yeast Two Hybrid Assay
Activates Transcription locally
Candidate Binding protein
Binds DNA
Bait- test protein
Reporter
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Single bait vector
Library of cDNAs in target vector
Already in yeast with Reporter genes
Transform into yeast
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lacZ for color HIS3 for growth
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REVERSE TWO HYBRID
Selection against reporter gene
allows Identification of altered proteins that no
longer interact Or third proteins or chemicals
that disrupt a protein-protein interaction
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All Protein 1 Fused to DNA Binding domain
Protein 1-96 Fused to Activation domain
Protein 1 Binds to Proteins 4, 7, 10
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3-HYBRID